Porphobilinogen (PBG) is a precursor to all biological tetrapyrroles (e.g. porphyrins, chlorins, corrins, F 430, phytochromes). PBG synthase (PBGS) catalyses the first common step in tetrapyrrole biosynthesis. PBGS is essential to all known life forms and is a principle target of the environmental toxin lead. Increased levels of the PBGS substrate 5-aminolevulinate (ALA) in lead poisoned individuals is believed to cause retardation in children and neurosis in adults. PBGS is a Zn(II) metalloenzyme whose inhibition by lead is a direct consequence of metal ion substitution. Our goal is to elucidate the catalytic mechanism of PBGS and to decipher the catalytic and structural role of Zn (II). PBGS catalyzes the only biological asymmetric condensation of identical gamma-keto, delta-amino acids, but is representative of larger classes of Zn-metalloenzymes and dehydratases. The PBGS reaction proceeds via a mechanism where the first ALA to bind forms a Schiff base between the ketonic carbon and an active site lysine. We have shown that Zn(II) and/or sulfhydryl groups are not required for Schiff base formation but are required for binding of the second ALA. Using 13C and 15 N NMR, we have identified 1) the enzyme-bound Schiff base as an imine (rather than an eneamine) of known stereochemistry and protonation states and 2) shown that enzyme-bound PBG contains a deprotonated amino group whose solution pKa is 11. The NMR studies have significantly advanced both our knowledge of the PBGS mechanism and the use of 13C and 15N NMR to observe protein-bound ligands. The remainder of the PBGS mechanism remains poorly characterized and is posed in the interrelated questions: 1) What are the tautomeric structures of enzyme-bound ALA? 2) Is the first bond formed between ALA molecules a C-C or C-N bond? 3) What is the activating role of Zn(II) and what steps are inhibited by lead? and 4) What are the functional active site amino acids? To answer these questions we are combining the techniques of chemical modification by affinity labelling, stable isotope labelling, and NMR, to determine the molecular structures which define the PBGS catalyzed reaction. We will also prepare analogs of two potential intermediate addition products and characterize their behavior as alternative substrates, reversible inhibitors, or affinity labels of PBGS. We will use probes of the intrinsic Zn(II) to determine if there are any interactions between the metal and the substrate(s), intermediates, or product. Complementary to our chemical modification studies, we will elucidate the amino acids present at the PBGS active site by purifying and sequencing the chemically modified peptides.